The agar disk diffusion test is the most widely used method in clinical laboratories, world-wide, for the evaluation of the ability of a growth-affecting substance (antimicrobial drugs or antibiotics) to inhibit the growth of infection-causing bacteria in samples obtained from body fluids or tissues of patients. The purpose of these tests is to select the drug for treatment most likely to facilitate control or eradication of the infection. With the disk diffusion test, a sample of the culture containing the organism to be tested is spread on the surface of an agar plate. Predetermined amounts of the various test drugs are contained, in powder form, in separate absorbent paper disks. The disks are placed on the surface of the agar previously inoculated with a test organism. During the subsequent period of incubation, the drug diffuses radially outward from the disk into the agar which forms a continuous radial gradient of drug concentration. At the same time, the organisms deposited on the surface utilize the nutrients provided in the agar to divide and, by virtue of their increasing numbers, to form a lawn of visible colonies in those areas of the plate where the presence of the drug does not inhibit their growth. Since inhibition is a function of drug concentration, the size of the inhibited area of growth, i.e. the circular region around the disk which is clear of visible colonies, becomes a measure of the ability of the drug to affect the growth of the test organism. The size of the zone of inhibition is thus used to characterize the degree of susceptibility or resistance of the test organism to a particular drug.
U.S. Pat. No. 4,514,495, which is assigned to the Assignee of the present invention, and a commercially available test known as the "E-Test", supplied by A. B. Biodisk, utilize visible inhibition zones to determine the effect of growth-affecting substances on the growth of microorganisms.
In the United Kingdom (and in some other countries) the decision as to the efficacy of a drug to inhibit growth of a test organism is made not in terms of the measured size of the zone of inhibition of the test organism. Instead, a comparison of the zone of inhibition of the test organism is made with the size of the zone produced by a reference organism having a response to the drug which is known. Such a comparison must be made under identical test conditions which requires that both the test and reference organisms be located on the surface of the same agar plate and subjected to the action of the drug from the same disk. This is accomplished by spreading the two organisms in separate but adjacent areas of the plate and placing the disk so that half of it is in the area of the reference organism and the other half in the area of the test organism. In other words, a line separates the two areas which bisects the disk. Two- half circle inhibition zones will be created which are compared in size to determine how the test organism responds relative to the known response of the reference organism. This method is known as the Stokes or the comparative disk diffusion test.
The comparative disk diffusion test can also be used to evaluate the effect of different concentrations of the same test organism on the ability of the drug to inhibit its growth. For example, some organisms produce a substance which inactivates the drug thereby making it less effective in its action against the organism. Higher concentrations of the organism being tested produce more of the inactivating substance and will create a smaller zone of inhibition. A comparative test in this case consists of using two different concentrations of the same organism to measure the relative size of the two-half circle zones produced from the same disk.
The comparative disk diffusion test, as currently practiced, requires that the disk be placed in a particular location on the agar plate so as to create the same gradient of drug concentrations in the two test areas. In addition, the comparison requires the measurement, or observation of two distinct zones of inhibition, in order to obtain the difference between them.
The prior art methods have the disadvantage of being predominately manually implemented. The deposition of the microorganism colonies is performed manually. Furthermore, it is necessary to accurately center the disk with respect to the boundary between two cultures. These manual steps can produce variation in the results. Furthermore, the manual deposition of the cultures does not permit a controllable gradient of the culture to be deposited on the culture medium. As a result, differential effects of the drug based upon different concentrations of the test organism may not be readily performed on the same culture medium.